Effect of common buffers and heterocyclic ligands on the binding of Cu(II) at the multimetal binding site in human serum albumin.

Sokołowska M, Pawlas K, Bal W - Bioinorg Chem Appl (2010)

Bottom Line:
The effects of warfarin and ibuprofen, specific ligands of hydrophobic pockets I and II in HSA on the Cu(II) binding to MBS were also investigated.The effects of ibuprofen were negligible, but warfarin diminished the MBS affinity for Cu(II) by a factor of 20, as a result of indirect conformational effects.These results indicate that metal binding properties of MBS can be modulated directly and indirectly by small molecules.

ABSTRACTVisible-range circular dichroism titrations were used to study Cu(II) binding properties of Multimetal Binding Site (MBS) of Human Serum Albumin (HSA). The formation of ternary MBS-Cu(II)-Buffer complexes at pH 7.4 was positively verified for sodium phosphate, Tris, and Hepes, the three most common biochemical buffers. The phosphate > Hepes > Tris order of affinities, together with strong spectral changes induced specifically by Tris, indicates the presence of both Buffer-Cu(II) and Buffer-HSA interactions. All complexes are strong enough to yield a nearly 100% ternary complex formation in 0.5 mM HSA dissolved in 100 mM solutions of respective buffers. The effects of warfarin and ibuprofen, specific ligands of hydrophobic pockets I and II in HSA on the Cu(II) binding to MBS were also investigated. The effects of ibuprofen were negligible, but warfarin diminished the MBS affinity for Cu(II) by a factor of 20, as a result of indirect conformational effects. These results indicate that metal binding properties of MBS can be modulated directly and indirectly by small molecules.

fig8: Comparison of titration curves for Cu(II) binding at MBS obtained in the presence of 1 mol equivalents of Ibu (experimental points, , and fits to the Kapp formula, ) and War (experimental points, , and fits to the Kapp formula, ).

Mentions:
Figure 8 presents titrations of HSA with Cu(II) ions in the presence of stoichiometric (1 mol equivalent) amounts of War and Ibu, performed in a 100 mM Hepes buffer, pH 7.4. The Ibu titration yielded the log Kapp value of 4.49(5), identical within the experimental error with that obtained in 100 mM Hepes in the absence of Ibu (4.45(5), Table 1). The presence of War had however a pronounced effect on Cu(II) binding. A very low log Kapp value of 3.2(2) was obtained. Therefore, the binding of War decreased the affinity of MBS for Cu(II) by a factor of 20. The Cu(II) binding at NTS was unaffected in both cases.

fig8: Comparison of titration curves for Cu(II) binding at MBS obtained in the presence of 1 mol equivalents of Ibu (experimental points, , and fits to the Kapp formula, ) and War (experimental points, , and fits to the Kapp formula, ).

Mentions:
Figure 8 presents titrations of HSA with Cu(II) ions in the presence of stoichiometric (1 mol equivalent) amounts of War and Ibu, performed in a 100 mM Hepes buffer, pH 7.4. The Ibu titration yielded the log Kapp value of 4.49(5), identical within the experimental error with that obtained in 100 mM Hepes in the absence of Ibu (4.45(5), Table 1). The presence of War had however a pronounced effect on Cu(II) binding. A very low log Kapp value of 3.2(2) was obtained. Therefore, the binding of War decreased the affinity of MBS for Cu(II) by a factor of 20. The Cu(II) binding at NTS was unaffected in both cases.

Bottom Line:
The effects of warfarin and ibuprofen, specific ligands of hydrophobic pockets I and II in HSA on the Cu(II) binding to MBS were also investigated.The effects of ibuprofen were negligible, but warfarin diminished the MBS affinity for Cu(II) by a factor of 20, as a result of indirect conformational effects.These results indicate that metal binding properties of MBS can be modulated directly and indirectly by small molecules.

ABSTRACTVisible-range circular dichroism titrations were used to study Cu(II) binding properties of Multimetal Binding Site (MBS) of Human Serum Albumin (HSA). The formation of ternary MBS-Cu(II)-Buffer complexes at pH 7.4 was positively verified for sodium phosphate, Tris, and Hepes, the three most common biochemical buffers. The phosphate > Hepes > Tris order of affinities, together with strong spectral changes induced specifically by Tris, indicates the presence of both Buffer-Cu(II) and Buffer-HSA interactions. All complexes are strong enough to yield a nearly 100% ternary complex formation in 0.5 mM HSA dissolved in 100 mM solutions of respective buffers. The effects of warfarin and ibuprofen, specific ligands of hydrophobic pockets I and II in HSA on the Cu(II) binding to MBS were also investigated. The effects of ibuprofen were negligible, but warfarin diminished the MBS affinity for Cu(II) by a factor of 20, as a result of indirect conformational effects. These results indicate that metal binding properties of MBS can be modulated directly and indirectly by small molecules.